Concern over the safety of inactivated or attenuated vaccines, in terms of administration to immunocompromised individuals and possible reversion to virulence has led to the development of subunit vaccines (composed of purified antigenic components of a micro-organism), DNA vaccines and also replication defective vaccine vectors. While these approaches are devoid of safety concerns, the purified proteins are generally less immunogenic than when expressed from a replicating organism. Furthermore, DNA vaccines and replication defective vaccine vectors encoding the gene of interest to stimulate the immune system generally express lower levels of antigen than replicating microorganisms. Thus multiple doses are required, which is uneconomic. For these vaccines to reach their full potential requires the incorporation of adjuvants to boost the immune response to the vaccine components.
Efficient stimulation of immune responses occurs when antigens are presented by potent professional antigen presenting cells. These consist primarily of DC's, present at the epithelial surfaces of the body, which form a surveillance system for recognition of foreign antigens. DC's are now recognized to take up and process antigens and present antigenic peptides in the context of Major Histocompatibility (MHC) molecules for recognition by T-lymphocytes (Banchereau et al 2000). However, besides recognition of foreign antigens, T lymphocytes require additional signals to become fully active (Bluestone, 1995). Costimulatory molecules (exemplified by CD80, CD86 and CD40) expressed on the surface of activated DC's provide these signals when they interact with their receptors on the T cell surface (Chambers C, 2001).
Replicating microorganisms (but not purified proteins or replication defective vaccine vectors) efficiently stimulate DC's to upregulate these costimulatory molecules and provide signals that induce strong immune responses. Thus it is now understood that one of the principal activities of adjuvants resides in their ability to mature DC, including upregulation of MHC class I and II and DC costimulatory molecules, which provide potent signals for the full activation of responding T (and also B) cells.
DCs are also recognized to play a fundamental role in determining the type of immune responses generated through the production of cytokines which regulate which T helper (Th) cell subset is primed (Lanzavecchia et al 2001). DC production of interleukins (IL-12, tumour necrosis factor alpha [TNF-α] and IL-4) is required to generate a balanced immune response. IL-12 and TNF-α production is critical in the generation of Th1 responses, the generation of CD8 T-cell responses, the production of IgG2a, IgG2b and IgG3 antibodies and recruitment of immune cells into the site of vaccine administration. IL-4 production is required to generate Th2 responses and stimulate B-cells to produce antibodies. Although IL-4 promotes Th2 responses it does not inhibit IL-12 in the generation of Th1 responses (Kalinski 1999). In the absence of IL-12, adjuvant induced production of IL-4 can result in not only IgG1 but also IgE which is associated with allergic disease (Hodge 2001) and serum IgA that can cause IgA nephropathy (Chintalacharuvu 2001).
Aluminium salts (alum) are the only universally licensed adjuvant for human use. However, they have the disadvantage of inducing allergic immunoglobulin-E (IgE) responses, and their widespread use in infant immunisation has been implicated in the recent rise in allergic diseases in the Western world. Although alum enhances antibody responses to vaccine components, it is not particularly effective at inducing cellular immunity, considered critical in control of intracellular pathogens such as HIV, Hepatitis C, Mycobacteria tuberculosis, Plasmodium falciparum or against tumours and is not a suitable adjuvant for mucosal (oral/nasal) immunisation.
While adjuvants such as Monophosphoryl lipid A (MPL), Quil-A, saponin and their derivatives (eg. QS7 and QS21) have been shown to elicit cellular immunity to vaccine components, their manufacture requires extensive extraction and purification procedures to remove toxicity and retain adjuvanticity, which reduce vaccine cost effectiveness. Moreover, the adjuvant activity of MPL formulations administered by mucosal routes is currently unproven in man.
While currently available vaccines primarily target the systemic immune system, most pathogens associated with global morbidity and mortality are transmitted across the mucosal surfaces of the body, initiating either localized infection (eg. rotavirus, the parainfluenza viruses and respiratory syncytial virus) or are disseminated from the mucosa to systemic tissues (eg. HIV, measles and Mycobacteria tuberculosis). Vaccines that can prevent pathogen dissemination from the initial site(s) of infection are likely to be more successful than those that target the blood or disease stage tissue or organs. However, the development of vaccines directed to the mucosal surfaces of the body has been stymied by the lack of adjuvants licensed for use at mucosal surfaces. Control of childhood infection with rotavirus, the parainfluenza viruses and respiratory synyctial virus, would significantly benefit by combining vaccine components with mucosal adjuvants which can be safely administered to infants below six months of age, without the concerns of allergic disease associated with the currently licensed aluminium salt based adjuvants.
The bacterial toxins, cholera toxin (CT) from Vibrio Cholerae and heat labile toxin (LT) from E.coli are powerful adjuvants for mucosal delivery in mice, though lower efficacy is reported in man. Concerns with their enterotoxicity preclude their use in man. Moreover a recent study had shown that CT can redirect vaccine proteins into olfactory tissues and may result in targeting of vaccine components to neuronal tissues (van Ginkel et al 2000). Thus, considerable effort has been directed towards altering the toxicity of CT and LT (Rappuoli et al 1999). Several groups have developed non toxic mutants of CT or LT, most of which have mutations in the CTA1-subunit that result in complete or partial disruption of the enzymatic activity associated with toxicity (reviewed Rappuoli et al 1999). However, all of these mutant holotoxins retain their promiscuous binding to the GM1-ganglioside receptor, leaving a potential ability to bind to all nucleated cells and so gain access to unwanted tissues such as the central nervous system (van Ginkel, et al 2000).
There thus remains a need for new effective systemic and mucosal adjuvants, suitable for human, veterinary, livestock and fisheries use which overcome the disadvantages of currently described compounds or substances with reported adjuvant activity.
There is also a need to provide greater protection against infectious disease for individuals with impaired immune systems (ie the young and the elderly). This is particularly relevant in the developing world, where reduced nutritional status is associated with significantly reduced antibody transfer from mother to child and in the elderly where there is a significant decline in immunity.
Infection in the neonatal, and perinatal period is primarily controlled via antibody acquired from the mother. IgG is transferred across the placenta (Lynden 2001 and Glezen 1999) and IgA from the mothers milk (Nagao 2001). This transfer of maternal antibody to foetus/infant is believed to play an important role in the protection of newborns against both bacterial and virus infections (Nagao 2001, Parissi-Crivelli 2000, Glezen 1999). Boosting the pregnant mother's antibody levels to antigens such as tetanus toxoid and influenza haemagglutinin (HA) has been shown to increase the protection of the foetus without harmful side effects to either mother or child (Glezen 1999). The ability to safely boost maternal antibodies in an antigen-independent manner, through materials, which can be used as a stand-alone immune potentiator, would be highly beneficial.
Elderly individuals demonstrate a reduced capacity to be successfully vaccinated (Ben-Yedidia et al 1998) with defects in their ability to mount protective immune responses (Fujihashi et al 2000). This is particularly important in stimulating protective immunity by annual vaccination against the new stains of influenza, which circulate and are a major cause of morbidity and mortality in the elderly. Both B and T cell responses are significantly decreased within the mucosal and systemic compartment with ageing (Fujihashi et al 2000). An aged related increase in IL-10 production (which can suppress DC maturation) and a decline in IL-12 production and dendritic cell antigen presentation, in the elderly, suggest functional defects in antigen presentation and T helper function (Spencer et al 1996, Tortorella et al 2002). Therefore, DC maturation factors, provided by adjuvants could provide the threshold level of co-stimulation required to initiate priming for vaccine induced T and B cell responses. Presently, alum is universally used an adjuvant, although its adjuvant properties are only minimally effective in the elderly when compared to the responses elicited by the same vaccine preparations used in young recipients (Davenport et al 1968). While several new adjuvants have been evaluated, studies demonstrate that (with the possible exception of MF59) these adjuvants have been ineffective in the elderly (Danenberg et al 1997).
Thus the development of a safe, non-reactogenic adjuvant which can enhance the immunogenicity of vaccines (eg against influenza virus, respiratory syncytial virus and the parainfluenza viruses) in the elderly is a high priority.
In accordance with the present invention, a composition for administration to a human or animal subject comprises spores of Bacillus subtilis (B. subtilis) in amount effective to stimulate or enhance immune responsiveness in the subject. Compositions according to the invention are capable of achieving either (a) an adjuvant effect, or (b) an immunomodulatory effect, or (c) an immune potentiation effect, or (d) a Dendritic cell maturation effect, or (e) any combination of two or more of effects (a) to (d). The ability of B. subtilis of spores to produce these varied effects is comprehensively demonstrated in the following description, especially in the section headed EXAMPLES.
The terms used above have connotations well-known to those skilled in the art. Thus, an adjuvant is a substance that enhances the immune response to an antigen with which it is administered. An immune modulator is a substance which changes the type (skews) the immune response without necessarily increasing it. An immune potentiator is a substance which increases or generates greater longevity of a pre-existing immune response. A dendritic cell maturator increases the ability of these cells to activate T cells.
Preferably the spores used for the purposes of the present invention are germination-deficient spores. These may be formulated either with antigenic materials, conveniently by simple admixture, or without such components, in which case the spores function alone as a cell maturator or activator of antigen-presenting cells, or as a non-specific booster of innate and humoral immunity, or as a booster of T independent and T dependent humoral immune responses. One such use of spores alone is for the preparation of a stand-alone vaccine either to boost waning immunity from a previous vaccination or as an immune booster for the elderly or for travellers; such a composition will consist essentially of spores and a suitable pharmaceutically and physiologically acceptable carrier or diluent.
Spores of B. subtilis may also be used in vitro to mature Dendritic cells and other antigen-presenting cells prior to administration of these e.g by returning isolated and treated cells to the patient. .
Bacillus subtilis: 
B. subtilis is a non-pathogenic Gram positive soil organism, which under conditions of nutrient deprivation forms spores, which are heat stable and resistant to UV radiation. For the last 30 years a commercial preparation of Bacillus spores (Enterogermina, Sanofi Winthrop, Milan Italy) has been licensed as a probiotic and reported to prevent or treat bacterial diarrhoea, though little was understood about the therapeutic effect. A few clinical reports have mentioned effects on the immune system associated with oral administration of the Enterogermina Bacillus spores obtained from the Sanofi Winthrop Company (Fiorini et al 1985, Cipriandi et al 1986a, Caruso et al 1993). These reports suggested that peripheral blood T-lymphocytes were activated following oral administration of Bacillus spores licensed as Enterogermina. In vitro studies have demonstrated that only the vegetative form and not the sporogenic form of the Enterogermina Bacillus obtained from the Sanofi-Winthrop Company (Cipriandi et al 1986b) induce lymphocyte activation. It was subsequently demonstrated that the spores present within the Enterogermina product germinate and colonize the intestinal tract (Mazza P, 1994). Current knowledge would thus ascribe the probiotic activity observed by Fiorini et al 1985, Cipriandi et al 1986b and Caruso et al 1993 with the vegetative cell, following germination of the orally delivered Enterogermina spore product.
Recently, a group in the UK reported that the probiotic preparations labelled as “B. subtilis”, currently available in Europe as Enterogermina (Sanofi-Winthrop) or Domuvar (Consorzio Farmaceutico e Biotechnologico Bioprogress a.r.i., Anagi-FR, Italy) and in S.E. Asia as Bactisubtil (Pharmaceutical Factory 24, Ho Chi Minh City, Vietnam), are mislabelled and in fact contain spores from Bacillus species which are taxonomically and phylogenetically unrelated to B. subtilis (Green et al 1999, Hoa et al 2000). The distinct taxonomic status of the Bacillus spores marketed as Enterogermina from B. subtilis has been confirmed (Senesi et al 2001). These authors also provide evidence that the Bacillus spores contained in commercial preparations of Enterogermina analysed from 1975 to the current date have remained genetically stable and are unequivocally members of the Bacillus alcalophilus subgroup, species B. clausii. 
There is a report suggesting that bacterial cells of B. subtilis and also an enzyme (Subtilisin) derived from the bacterial cell of B. subtilis (of unknown taxonomic status) augment cutaneous anaphylaxis, a serious immunopathologic reaction (Malkiel et al 1971). However, the spores were reported to be ineffective (Malkiel et al 1971). A protein component termed Protodyne reported as common to several genera of Gram-negative and Gram-positive bacteria (including B. subtilis) derived from the bacterial protoplasm was described to exert an immunostimulatory activity (Houba et al 1992). Contemporary knowledge may ascribe Protodyne as peptidoglycan-techoic acid, derived from the cell wall of many bacterial cells (including B. subtilis vegetative cells) which is weakly pyrogenic and mitogenic (Hinmanen et al 1993).
In summary, there are conflicting probiotic and immunopathologic reports attributed to the vegetative cells of B. subtilis (and other genera of Bacilli) without documentation of any adjuvanticity, B cell or DC immunopotentiatory or immunomodulatory or DC maturation activity associated with the sporegenic form.
The invention uses spores of B. subtilis subspecies subtilis (also known as Ehrenberg 1835 or Cohn 1872); defined as all Bacillus strains which contain a 16SrRNA [small subunit ribosomal RNA] 99% similar at the genetic level to the B. subtilis strain 168 [ATCC number 23857] (Maidak et al, 2000), where the biochemical, metabolic and morphological properties of currently known Bacillus strains are defined by Bergey's Manual of Systematic Bacteriology, section 13, pp 1104-1139. Examples of B. subtilis strains with 99% or higher 16S rRNA genetic similarity includes ATCC numbers; 9799, 35148, 15563, 33234, 55567.
The invention uses spores from B. subtilis as an adjuvant by simply admixture with a candidate vaccine to boost the immune response to the vaccine components
Attributes of B. Subtilis Spores:
The spores are extremely cheap to produce, do not require purification procedures, are heat and UV stable, and therefore do not require cold chain storage. They are suitable for incorporation into pharmaceutical compositions for administration as suspensions or from a lyophilised form, thus affording indefinite storage, and are suitable for systemic or mucosal delivery e.g by intravenous, intradermal, intramuscular, subcutaneous, transdermal or intraperitoneal administration or by oral, nasal, respiratory, pulmonary topical, gill delivery, intrathecal, buccal (sublingal), rectal or vaginal delivery. The dose at which the substance of the invention is administered to a patient or to an animal will depend upon a variety of factors such as the age, weight and general condition of the patient, the condition that is being treated and the route by which the substance is being administered. A suitable dose may however be 105 to 1011 spores per dose. If doses in this range are not sufficient, the dose may be increased. Those skilled in the art will recognise the appropriate dosage level to test, from research reported herein. Without intending any limitation as to course of treatment, the adjuvant, immunemodulator, immune potentiator or dendritic cell-maturating agent/innate stimulator could be administered on a number of occasions.
B. subtilis is classified by the term, GRAS (generally regarded as safe) organism, thus germination competent spores can be used. For certain applications it will be desirable to use germination deficient spores, which can also be administered to individuals with immunocompromised immune systems, where the adjuvant activity of the spore may safely augment the immunogenicity of a vaccine. This has particular application to childhood vaccination, particularly in the first year of life and also to individuals with acute/chronic acquired immunodeficiency, through infection with HIV, measles, or Mycobacteria tuberculosis. The use of germination deficient spores provides additional benefit, in veterinary and commercial livestock, poultry or fish vaccine applications, where there are concerns of antibiotic resistance transfer to man associated with live bacterial vectors or probiotics used in oral bacteriotherapy.
Germination deficient spores can be generated by heat inactivation or by inactivation of B. subtilis genes, examples of which include the following list of genes (followed by their accession number): gerAA P07868, gerAB P07869, gerAC P07870, gerBA P39569, gerBB P39570, gerBC P39571, gerD 555 P16450, gerE 222 P11470, gerKA P49939, gerKB P49940, gerKC P49941, gerM P39072.
The present invention is to be distinguished from the use of B. subtilis spores as a vaccine vector in which antigenic sequences are cloned and expressed i.e as an epitope presentation system.